The long-term goal of this proposal is to understand the molecular and physiological bases of cold nociception. Thermosensory nociception is a specialized form of somatosensation essential to the survival of all metazoans. Thermosensory nociception alerts the organism to potential environmental dangers coupled with pain sensation thereby serving as a protective mechanism for driving adaptive behavioral responses to safeguard against incipient damage. Despite this importance, the fundamental molecular and biophysical bases of cold nociception remain poorly understood. Molecularly, transient receptor potential channels (i.e. thermoTRPs) play critical roles in thermosensation, however, relatively less is known regarding how thermoTRPs mechanistically function in regulating noxious cold detection. Neurologically, acute and chronic pain may manifest as altered thermosensory nociception whereby innocuous thermal stimuli erroneously engage nociceptive circuitry leading to neuropathic pain. Cold hypersensitivity is associated with multiple sclerosis, fibromyalgia, stroke, and chemotherapy-induced neuropathy resulting in neuropathic pain, however the mechanisms underlying cold sensitization are largely unknown. Here, we will investigate a fundamental problem of how multimodal sensory neurons discriminately detect noxious cold stimuli to elicit nocict9ptive behavior using Drosophila as a model system in combination with bi- directionally linked neurogenetic, neurogenomic, cellular imaging, electrophysiological, behavioral, computational modeling, and bifurcation analyses. We aim to uncover molecular and biophysical bases for cold-evoked nociceptive stimulus coding, including the functional properties of thermoTRPs and Ca2 signaling dynamics in this process. The project aims and outcomes of this research will significantly advance our knowledge of cold nociception by addressing three open questions: (1) What are the molecular and biophysical bases of cold nociceptive stimulus coding? (2) How do multimodal nociceptive neurons discriminately detect noxious stimuli (e.g. cold) to drive nocifensive behavior? (3) How do thermoTRPs and Ca2 signaling mechanisms mechanistically function in regulating noxious cold detection? More generally, the bi-directional integration of experimental and computational approaches in a closed- loop investigational strategy is well-suited to transform our understanding of cold nociception by elucidating potentially generalizable mechanisms of cold thermosensory coding, including roles of TRP channels and. Ca2 homeostasis in sensory-evoked neural activity. RELEVANCE (See instructions): The perception of noxious stimuli is often coupled to pain sensation as a protective mechanism, however altered temperature sensation may lead to neuropathic pain (e.g. in multiple sclerosis, fibromyalgia, and stroke) where patients experience pain due to cold hypersensitivity. By uncovering basic mechanisms of noxious cold perception, we develop important insights on neural integration of painful stimuli providing potential routes for understanding and treating neurological disease when this process is disrupted.